JP6834129B2 - Separator and column - Google Patents

Description

The present invention relates to a separating material and a column.

Conventionally, when biopolymers typified by proteins are separated and purified, generally, an ion exchanger based on a porous synthetic polymer and an ion exchanger based on a crosslinked gel of a hydrophilic natural polymer are used. Etc. are used. In the case of an ion exchanger based on a porous synthetic polymer, the volume change due to salt concentration is small, so when it is packed in a column and used for chromatography, it tends to have excellent pressure resistance during liquid passage. However, when this ion exchanger is used for separating proteins and the like, non-specific adsorption such as irreversible adsorption based on hydrophobic interaction occurs, so peak asymmetry occurs, or ion exchange occurs due to the hydrophobic interaction. There is a problem that the protein adsorbed on the body cannot be recovered while being adsorbed.

On the other hand, in the case of an ion exchanger whose parent is a crosslinked gel of a hydrophilic natural polymer typified by a polysaccharide such as dextran or agarose, there is an advantage that there is almost no non-specific adsorption of proteins. However, this ion exchanger has a drawback that it swells remarkably in an aqueous solution, the volume change due to the ionic strength of the solution and the volume change between the free acid type and the loaded type are large, and the mechanical strength is not sufficient. In particular, when the crosslinked gel is used for chromatography, there is a drawback that the pressure loss during liquid passage is large and the gel is consolidated by liquid passage.

In order to overcome the drawbacks of crosslinked gels of hydrophilic natural polymers, for example, a complex in which a gel such as a natural polymer gel is held in the pores of a porous polymer is known in the field of peptide synthesis ( For example, see Patent Document 1). By using such a complex, the loading coefficient of the reactive substance is increased, and high-yield synthesis becomes possible. Further, since the gel is surrounded by a hard synthetic polymer substance, there is an advantage that the volume does not change and the pressure of the flow-through passing through the column does not change even when used in the form of a column bed.

A separating material in which an inorganic porous body such as Celite holds a xerogel such as a polysaccharide such as dextran or cellulose is known (see, for example, Patent Documents 2 and 3). Diethylaminoethyl (DEAE) groups and the like are added to this gel in order to add adsorption performance, and it is used for removing hemoglobin. Such a separating material has good liquid permeability in a column.

An ion exchanger of a hybrid copolymer in which the pores of a copolymer having a macronetwork structure are filled with a crosslinked copolymer gel synthesized from a monoma is known (see, for example, Patent Document 4). When the degree of cross-linking is low, the cross-linked copolymer gel has problems such as pressure loss and volume change, but by using a hybrid copolymer, the liquid passage characteristics are improved, the pressure loss is small, and the ion exchange capacity is improved. Leak behavior is improved.

Further, a composite filler in which a crosslinked gel of a hydrophilic natural polymer having a huge network structure is filled in the pores of an organic synthetic polymer substrate has been proposed (see, for example, Patent Documents 5 and 6). Further, a technique for synthesizing porous particles composed of glycidyl methacrylate and an acrylic crosslinked monoma is known (see, for example, Patent Document 7).

U.S. Pat. No. 4,965,289 U.S. Pat. No. 4,335,017 U.S. Pat. No. 4,336,161 U.S. Pat. No. 3,966,489 Japanese Unexamined Patent Publication No. 1-254247 U.S. Pat. No. 5,114,577 Japanese Unexamined Patent Publication No. 2009-244067

However, the conventional separating material has a problem that the protein is often non-specifically adsorbed, the adsorbed amount is not sufficient, and the liquid permeability is poor when used as a column.

Therefore, an object of the present invention is to provide a separating material in which non-specific adsorption of a protein is reduced, the amount of adsorption is high, and the liquid permeability is excellent when used as a column, and a column provided with the separating material.

The present invention provides the separating material according to the following [1] to [9] and a column including the separating material.
[1] A graft polymer comprising porous polymer particles and a coating layer that covers at least a part of the surface of the porous polymer particles, and the coating layer is a graft polymer in which dextran is grafted on a crosslinked polymer having a hydroxyl group. Including, separating material.
[2] The separating material according to [1], wherein the porous polymer particles contain a polymer having a structural unit derived from a monoma containing divinylbenzene.
[3] The separating material according to [1] or [2], which has a pore diameter (mode diameter) of 0.1 to 0.5 μm.
[4] The separating material according to any one of [1] to [3], wherein the coefficient of variation of the particle size of the porous polymer particles is 5 to 15%.
[5] The separating material according to any one of [1] to [4], wherein the crosslinked polymer having a hydroxyl group is a crosslinked polymer derived from a polysaccharide or a modified product thereof.
[6] The separating material according to any one of [1] to [5], wherein the crosslinked polymer having a hydroxyl group is a crosslinked polymer derived from agarose or a modified product thereof.
[7] The separating material according to any one of [1] to [6], wherein the weight average molecular weight of dextran is 40,000 to 1,000,000.
[8] The separating material according to any one of [1] to [7], which comprises a coating layer of 30 to 400 mg per 1 g of porous polymer particles.
[9] A column comprising the separating material according to any one of [1] to [8].

According to the present invention, it is possible to provide a separating material having reduced nonspecific adsorption of a protein, a high adsorption amount, and excellent liquid permeability when used as a column, and a column provided with the separating material.

Hereinafter, preferred embodiments of the present invention will be described, but the present invention is not limited to these embodiments.
In the numerical range indicated by using "~" in the present specification, the numerical values before and after "~" are included as the minimum value and the maximum value, respectively.

<Separation material>
The separating material of the present embodiment includes porous polymer particles and a coating layer that covers at least a part of the surface of the porous polymer particles. In the present specification, the “surface of the porous polymer particles” includes not only the outer surface of the porous polymer particles but also the surface of the pores inside the porous polymer particles. Further, in the present specification, (meth) acrylic acid means acrylic acid or methacrylic acid, and the same applies to other similar expressions such as (meth) acrylate.

(Porous polymer particles)
The porous polymer particles according to the present embodiment are particles containing a polymer obtained by polymerizing a monoma in the presence of a porosifying agent, and can be synthesized by, for example, conventional suspension polymerization, emulsion polymerization, or the like. .. That is, the porous polymer particles include, for example, a polymer having a structural unit derived from the monoma. The monoma is not particularly limited, and for example, an acrylic monoma or a styrene-based monoma can be used. Specific examples thereof include the following polyfunctional monomas and monofunctional monomas.

Examples of the polyfunctional monoma include divinyl compounds such as divinylbenzene, divinylbiphenyl, divinylnaphthalene, and divinylphenanthrene; (poly) ethylene glycol di (meth) acrylate, (poly) propylene glycol di (meth) acrylate, and (poly). (Poly) alkylene glycol-based di (meth) acrylates such as tetramethylene glycol di (meth) acrylates; trimethylolpropane tri (meth) acrylates, tetramethylolmethane tri (meth) acrylates, tetramethylolpropane tetra (meth) acrylates, penta. Trifunctional or higher functional (meth) acrylates such as erithitol tri (meth) acrylate, 1,1,1-trishydroxymethylethanetri (meth) acrylate, 1,1,1-trishydroxymethylpropanetriacrylate; ethoxylated bisphenol A Di (meth) acrylate, propoxylated bisphenol A di (meth) acrylate, tricyclodecanedimethanol di (meth) acrylate, 1,1,1-trishydroxymethylethanedi (meth) acrylate, cyclohexane ethoxylated Examples thereof include di (meth) acrylates such as dimethanol di (meth) acrylate; diallyl phthalate and its isomers; trimethylolisocyanurate and its derivatives. Among these monomas, for example, NK ester (A-TMPT-6P0, A-TMPT-3E0, A-TMM-3LMN, A-GLY series, A-9300, AD-TMP, AD) manufactured by Shin-Nakamura Chemical Industry Co., Ltd. -TMP-4CL, ATM-4E, A-DPH) and the like are commercially available. These polyfunctional monomas can be used alone or in combination of two or more. Among the above, it is preferable that the monoma contains divinylbenzene from the viewpoint of durability, acid resistance and alkali resistance. That is, the porous polymer particles preferably contain a polymer having a structural unit derived from a monoma containing divinylbenzene.

When divinylbenzene is contained as a monoma unit, the porous polymer particles preferably contain divinylbenzene in an amount of 60% by mass or more, more preferably 70% by mass or more, and more preferably 80% by mass or more based on the total mass of monoma. More preferred. It is preferable to contain divinylbenzene in an amount of 60% by mass or more because the alkali resistance becomes better.

Examples of the monofunctional monoma include styrene, o-methylstyrene, m-methylstyrene, p-methylstyrene, α-methylstyrene, o-ethylstyrene, m-ethylstyrene, p-ethylstyrene, 2,4-. Dimethylstyrene, pn-butylstyrene, pt-butylstyrene, pn-hexylstyrene, pn-octylstyrene, pn-nonylstyrene, pn-decylstyrene, pn-dodecyl Styrene such as styrene, p-methoxystyrene, p-phenylstyrene, p-chlorostyrene, 3,4-dichlorostyrene and derivatives thereof; methyl acrylate, ethyl acrylate, propyl acrylate, n-butyl acrylate, acrylic acid Isobutyl, hexyl acrylate, 2-ethylhexyl acrylate, n-octyl acrylate, dodecyl acrylate, lauryl acrylate, stearyl acrylate, 2-chloroethyl acrylate, phenyl acrylate, methyl α-chloroacrylate, methyl methacrylate , Ethyl methacrylate, propyl methacrylate, n-butyl methacrylate, isobutyl methacrylate, hexyl methacrylate, 2-ethylhexyl methacrylate, n-octyl methacrylate, dodecyl methacrylate, lauryl methacrylate, stearyl methacrylate, etc. (meth) ) Acrylic acid ester; vinyl ester such as vinyl acetate, vinyl propionate, vinyl benzoate, vinyl butyrate; N-vinyl compound such as N-vinylpyrrole, N-vinylcarbazole, N-vinylindole, N-vinylpyrrolidone; Fluorinated monomas such as vinyl oxide, vinylidene fluoride, tetrafluoroethylene, hexafluoropropylene, trifluoroethyl acrylate, tetrafluoropropyl acrylate; and conjugated diene such as butadiene and isoprene. These monofunctional monomas may be used alone or in combination of two or more. Among the above, it is preferable to use styrene from the viewpoint of excellent acid resistance and alkali resistance. Further, a styrene derivative having a functional group such as a carboxy group, an amino group, a hydroxyl group or an aldehyde group can also be used.

Examples of the porosifying agent include aliphatic or aromatic hydrocarbons, esters, ketones, ethers, alcohols, etc., which are organic solvents that promote phase separation during polymerization and promote porosification of particles. Be done. Specific examples of the porosifying agent include toluene, xylene, cyclohexane, octane, butyl acetate, dibutyl phthalate, methyl ethyl ketone, dibutyl ether, 1-hexanol, 2-octanol, decanol, lauryl alcohol, cyclohexanol and the like. .. These porosifying agents may be used alone or in combination of two or more.

The porosifying agent can be used, for example, in a range of more than 0 and 200% by mass or less with respect to the total mass of the monoma. The porosity of the porous polymer particles can be controlled by the amount of the porosity agent. Furthermore, the size and shape of the pores of the porous polymer particles can be controlled by the type of the porosifying agent.

Water used as a solvent can also be used as a porosifying agent. When water is used as a porosifying agent, the particles can be made porous by dissolving an oil-soluble surfactant in a monoma and absorbing water.

Examples of the oil-soluble surfactant used for porosification include sorbitan monoesters of branched C16 to C24 fatty acids, chain unsaturated C16 to C22 fatty acids, and chain saturated C12 to C14 fatty acids, such as sorbitan monolaurate and sorbitan. Solbitan monoester derived from monooleate, sorbitan monomillistate or coconut fatty acid; diglycerol monoester of branched C16-C24 fatty acid, chain unsaturated C16-C22 fatty acid or chain saturated C12-C14 fatty acid, eg di Diglycerol monoester of glycerol monooleate (eg, C18: 1 (18 carbons, 1 double bond) fatty acid), diglycerol monomillistate, diglycerol monoisostearate or diglycerol mono of coconut fatty acid Esters; diglycerol monofatty ethers of branched C16-C24 alcohols (eg, gelve alcohol), chain unsaturated C16-C22 alcohols or chain saturated C12-C14 alcohols (eg palm fatty alcohols), and mixtures thereof. Can be mentioned.

Of these, sorbitan monolaurate (eg, SPAN® 20, purity preferably greater than about 40%, more preferably greater than about 50%, even more preferably greater than about 70% sorbitan monolaurate). Rate), sorbitan monooleate (eg, SPAN® 80, purity preferably greater than about 40%, more preferably greater than about 50%, even more preferably greater than about 70% sorbitan monooleate. ), Diglycerol monooleate (eg, diglycerol monooleate having a purity of preferably greater than about 40%, more preferably greater than about 50%, even more preferably greater than about 70%), diglycerol monoisostearate. (For example, diglycerol monoisostearate having a purity of preferably greater than about 40%, more preferably greater than about 50%, even more preferably greater than about 70%), diglycerol monomillistate (purity preferably greater than about 40). More than%, more preferably more than about 50%, even more preferably more than about 70% sorbitan monomillistate), diglycerol cocoyl (eg, lauryl group, myristyl group, etc.) ether, or mixtures thereof.

These oil-soluble surfactants are preferably used in the range of 5 to 80% by mass with respect to the total mass of the monoma. When the content of the oil-soluble surfactant is 5% by mass or more, the stability of the water droplet is sufficient, and it becomes difficult to form a large single pore. Further, when the content of the soluble surfactant is 80% by mass or less, the porous polymer particles are more likely to retain their shape after polymerization.

Examples of the aqueous medium used in the polymerization reaction include water, a mixed medium of water and a water-soluble solvent (for example, a lower alcohol), and the like. The aqueous medium may contain a surfactant. As the surfactant, any of anionic, cationic, nonionic and zwitterionic surfactants can be used.

Examples of the anionic surfactant include fatty acid oils such as sodium oleate and potassium castor oil, alkyl sulfates such as sodium lauryl sulfate and ammonium lauryl sulfate, alkylbenzene sulfonates such as sodium dodecylbenzene sulfonate, and alkylnaphthalene sulfates. Dialkyl sulfosuccinates such as acid salts, alkane sulfonates, sodium dioctyl sulfosuccinate, alkernyl succinate (dipotassium salt), alkyl phosphate ester salts, naphthalene sulfonate formalin condensates, polyoxyethylene alkyl phenyl ether sulfate Examples thereof include salts, polyoxyethylene alkyl ether sulfates such as sodium polyoxyethylene lauryl ether sulfate, and polyoxyethylene alkyl sulfates.

Examples of the cationic surfactant include alkylamine salts such as laurylamine acetate and stearylamine acetate, and quaternary ammonium salts such as lauryltrimethylammonium chloride.

Examples of nonionic surfactants include hydrocarbon-based nonionic surfactants such as polyethylene glycol alkyl ethers, polyethylene glycol alkylaryl ethers, polyethylene glycol esters, polyethylene glycol sorbitan esters, polyalkylene glycol alkylamines, and amides. Examples thereof include polyethylene oxide adducts of silicon, polyether-modified silicon nonionic surfactants such as polypropylene oxide adducts, and fluorine nonionic surfactants such as perfluoroalkyl glycols.

Examples of the amphoteric ion-based surfactant include hydrocarbon surfactants such as lauryldimethylamine oxide, phosphate ester-based surfactants, and phosphite ester-based surfactants.

As the surfactant, one type may be used alone or two or more types may be used in combination. Among the above-mentioned surfactants, anionic surfactants are preferable from the viewpoint of dispersion stability during polymerization of monomas.

Examples of the polymerization initiator added as needed include benzoyl peroxide, lauroyl peroxide, orthochlorobenzoyl peroxide, benzoyl orthomethoxy peroxide, 3,5,5-trimethylhexanoyl peroxide, and tert-butylper. Organic peroxides such as oxy-2-ethylhexanoate, di-tert-butyl peroxide; 2,2'-azobisisobutyronitrile, 1,1'-azobiscyclohexanecarbonitrile, 2,2' Examples thereof include azo compounds such as −azobis (2,4-dimethylvaleronitrile). The polymerization initiator can be used in the range of 0.1 to 7.0 parts by mass with respect to 100 parts by mass of the monoma, for example.

The polymerization temperature can be appropriately selected depending on the type of the monoma and the polymerization initiator. The polymerization temperature is preferably 25 to 110 ° C, more preferably 50 to 100 ° C.

In the synthesis of porous polymer particles, a polymer dispersion stabilizer may be added in order to improve the dispersion stability of the particles.

Examples of the polymer dispersion stabilizer include polyvinyl alcohol, polycarboxylic acid, celluloses (hydroxyethyl cellulose, carboxymethyl cellulose, methyl cellulose, etc.), polyvinylpyrrolidone, and an inorganic water-soluble polymer compound such as sodium tripolyphosphate is also used in combination. can do. Of these, polyvinyl alcohol or polyvinylpyrrolidone is preferable. The amount of the polymer dispersion stabilizer added is preferably 1 to 10 parts by mass with respect to 100 parts by mass of the monoma.

In order to prevent the monoma from emulsion polymerization in water, water-soluble polymerization inhibitors such as nitrites, sulfites, hydroquinones, ascorbic acids, water-soluble B vitamins, citric acid, and polyphenols may be used. ..

The average particle size of the porous polymer particles is preferably 500 μm or less, more preferably 300 μm or less, still more preferably 100 μm or less. The average particle size of the porous polymer particles is preferably 10 μm or more, more preferably 30 μm or more, and further preferably 50 μm or more. When the average particle size of the porous polymer particles is 10 μm or more, the column pressure after column filling tends to be suppressed.

The coefficient of variation (CV) of the particle size of the porous polymer particles is preferably 5 to 15%, more preferably 5 to 14%, and 5 to 5% from the viewpoint of improving the liquid permeability. It is more preferably 13%. C. V. As a method of reducing the amount of waste, it is possible to monodisperse with an emulsifying device such as a micro process server (manufactured by Hitachi, Ltd.).

C. of the average particle size and particle size of the porous polymer particles or separating material. V. Can be obtained by the following measurement method.
1) Disperse the porous polymer particles or separating material in water (including a dispersant such as a surfactant) using an ultrasonic disperser, and a dispersion containing 1% by mass of the porous polymer particles or separating material. To prepare.
2) Using a particle size distribution meter (Sysmex Flow, manufactured by Sysmex Corporation), images of about 10,000 porous polymer particles or separating materials in the dispersion liquid show the average particle size and particle size of C.I. V. To measure.

The porosity (pore volume) of the porous polymer particles is preferably 30% by volume or more and 70% by volume or less based on the total volume of the porous polymer particles, and more preferably 40% by volume or more and 70% by volume or less. preferable. The porous polymer particles preferably have macropores (macropores), and the pore diameter (mode diameter) of the porous polymer particles is preferably 0.1 μm or more and 0.5 μm or less. The pore size of the porous polymer particles is more preferably 0.2 μm or more and 0.5 μm or less. When the pore diameter is 0.1 μm or more, a substance tends to easily enter the pores, and when the pore diameter is 0.5 μm or less, the specific surface area becomes sufficient. These can be adjusted by the above-mentioned porosifying agent.

The specific surface area of the porous polymer particles ^{is preferably 30 m 2} / g or more. From the viewpoint of higher practicality, the specific surface area is ^{more preferably 35 m 2} / g or more, and further preferably 40 m ² / g or more. When the specific surface area is 30 m ² / g or more, the amount of the substance to be separated tends to be large.

The pore diameter (mode diameter), specific surface area and porosity of the porous polymer particles or the separating material can be measured by using a mercury intrusion measuring device (Autopore: manufactured by Shimadzu Corporation), for example, as follows. Approximately 0.05 g of a sample is added to a standard 5 mL powder cell (stem volume 0.4 mL) and measured under the condition of an initial pressure of 21 kPa (approximately 3 psia, equivalent to a pore diameter of approximately 60 μm). The mercury parameters are set to the device default mercury contact angle of 130 degrees and mercury surface tension of 485 days / cm. Further, each value is calculated by limiting the pore diameter to the range of 0 to 5 μm.

(Coating layer)
The coating layer according to the present embodiment contains a graft polymer in which dextran is grafted on a crosslinked polymer having a hydroxyl group. By coating the porous polymer particles with such a coating layer, it is possible to suppress the improvement of the column pressure, the non-specific adsorption of the protein, and the protein adsorption of the separating material. The amount can be improved.

Examples of the graft polymer include a graft polymer obtained by introducing a group capable of reacting with dextran into a crosslinked polymer having a hydroxyl group and reacting the group with dextran.

The crosslinked polymer having a hydroxyl group preferably has two or more hydroxyl groups in one molecule, and more preferably a hydrophilic polymer. Examples of the crosslinked polymer having a hydroxyl group include a crosslinked polymer derived from a polysaccharide or a modified product thereof, a crosslinked polymer derived from polyvinyl alcohol or a modified product thereof, and the like. Examples of the polysaccharide include agarose, dextran, cellulose, chitosan and the like. From the viewpoint of further reducing non-specific adsorption and further improving the protein adsorption performance, the crosslinked polymer having a hydroxyl group is preferably a crosslinked polymer derived from a polysaccharide or a modified product thereof, and is derived from agarose or a modified product thereof. It is more preferable that it is a crosslinked polymer of.

Further, the crosslinked polymer having a hydroxyl group may be a modified product modified by a hydrophobic group from the viewpoint of improving the interfacial adsorption ability. Examples of the hydrophobic group include an alkyl group having 1 to 6 carbon atoms and an aryl group having 6 to 10 carbon atoms. Examples of the alkyl group having 1 to 6 carbon atoms include a methyl group, an ethyl group, a propyl group and the like. Examples of the aryl group having 6 to 10 carbon atoms include a phenyl group and a naphthyl group. A hydrophobic group is introduced by reacting a functional group (for example, an epoxy group) that reacts with a hydroxyl group and a compound having a hydrophobic group (for example, glycidyl phenyl ether) with a polymer having a hydroxyl group by a conventionally known method. Can be done.

Examples of the group capable of reacting with dextran include a group capable of reacting with a hydroxyl group of dextran and a group capable of reacting with an aldehyde group having a reducing end of dextran. Examples of the group capable of reacting with the hydroxyl group include an epoxy group, a vinyl sulfone group, and an isocyanate. Examples of the group capable of reacting with the aldehyde group include an amino group. From the viewpoint of further reducing non-specific adsorption, the group capable of reacting with dextran is preferably an epoxy group.

The weight average molecular weight (Mw) of dextran grafted on the crosslinked polymer having a hydroxyl group is preferably 40,000 to 1,000,000, more preferably 40,000 to 750000, and even more preferably 40,000 to 500,000. When Mw is 1,000,000 or less, the possibility that the pores of the porous polymer particles are blocked is reduced. When Mw is 40,000 or more, the protein adsorption ability is further improved.

In the present specification, Mw refers to a numerical value measured by GPC using pullulan having a different molecular weight as a standard substance and using water as a solvent.

The mass of dextran grafted on the crosslinked polymer having a hydroxyl group is preferably 1/5 or less, more preferably 1/7 or less, and 1/7 or less, based on the mass of the crosslinked polymer having a hydroxyl group. It is more preferably 10 or less.

The amount of dextran grafted onto the crosslinked polymer having a hydroxyl group can be calculated, for example, by thermogravimetric analysis before and after grafting.

(Method of forming the coating layer)
For the coating layer, for example, a polymer having a hydroxyl group is adsorbed on the surface of the porous polymer particles, and then the polymer is crosslinked to form a layer of a crosslinked polymer having a hydroxyl group on the surface of the porous polymer particles. Further, it can be formed by a method of grafting dextran to the crosslinked polymer.

Hereinafter, a specific example of the method for forming the coating layer will be described.

(Adsorption process)
First, a solution of a polymer having a hydroxyl group is adsorbed on the surface of the porous polymer particles.

Examples of the polymer having a hydroxyl group include polysaccharides or modified products thereof, polyvinyl alcohol or modified products thereof. Examples of the polysaccharide include agarose, dextran, cellulose, chitosan and the like. From the viewpoint of further reducing non-specific adsorption, the Mw of the polymer having a hydroxyl group is preferably 1000 to 20000, more preferably 1000 to 150,000, and even more preferably 1000 to 120,000.

The solvent for the solution of the polymer having a hydroxyl group is not particularly limited as long as it can dissolve the polymer having a hydroxyl group, but water is the most common. The concentration of the polymer dissolved in the solvent is preferably 5 to 20 (mg / mL). The solution is impregnated with the porous body in order to adsorb the polymer having a hydroxyl group on the surface of the porous body (including the inside of the pores). In the impregnation method, porous polymer particles are added to a polymer solution having a hydroxyl group and stirred for a certain period of time. The impregnation time varies depending on the surface condition of the porous body, but usually, after stirring for 6 to 12 hours, the polymer concentration becomes an equilibrium state with the external concentration inside the porous body. Then, it is washed with a solvent such as water and alcohol to remove the polymer having an unadsorbed hydroxyl group.

(Crosslinking)
Next, a cross-linking agent is added to cause a cross-linking reaction of the polymer having a hydroxyl group adsorbed on the surface of the porous polymer particles to form a cross-linked body. At this time, the crosslinked body has a three-dimensional crosslinked network structure having a hydroxyl group.

Examples of the cross-linking agent include functional groups active on hydroxyl groups, such as epihalohydrin such as divinyl sulfone and epichlorohydrin, dialdehyde compound such as glutaraldehyde, diisocyanate compound such as methylene diisocyanate, and glycidyl compound such as ethylene glycol diglycidyl ether. Examples thereof include compounds having two or more of. Further, when a compound having an amino group such as chitosan is used as the polymer having a hydroxyl group, dihalide such as dichlorooctane can also be used as a cross-linking agent.

A catalyst is usually used for this cross-linking reaction. As the catalyst, conventionally known ones can be appropriately used according to the type of the cross-linking agent. For example, when the cross-linking agent is epichlorohydrin, ethylene glycol diglycidyl ether or the like, an alkali such as sodium hydroxide is effective. In the case of dialdehyde compounds, mineral acids such as hydrochloric acid are effective.

The cross-linking reaction with a cross-linking agent is usually carried out by adding a cross-linking agent to a system in which porous polymer particles impregnated with a polymer solution having a hydroxyl group or the like in the pores are dispersed and suspended in an appropriate medium. Will be done. When a polysaccharide is used as a polymer having a hydroxyl group, the amount of the cross-linking agent added is within the range of 0.1 to 100 mol times, assuming that one unit of the monosaccharide is 1 mol, and the desired separation is performed. It may be selected according to the performance of the material. In general, when the amount of the cross-linking agent added is small, the coating layer tends to be easily peeled off from the porous polymer particles. Further, when the amount of the cross-linking agent added is excessive and the reaction rate with the polymer having a hydroxyl group is high, the characteristics of the polymer having a hydroxyl group as a raw material tend to be impaired.

The amount of the catalyst used varies depending on the type of cross-linking agent, but usually, when a polysaccharide is used as a polymer having a hydroxyl group, it is assumed that one unit of the monosaccharide forming the polysaccharide is 1 mol. It is preferably used in the range of 0.01 to 10 mol times, more preferably 0.1 to 5 mol times.

For example, when the cross-linking reaction condition is a temperature condition, the temperature of the reaction system is raised, and when the temperature reaches the reaction temperature, a cross-linking reaction occurs.

As a medium for dispersing and suspending porous polymer particles impregnated with a polymer solution having a hydroxyl group, the polymer, a cross-linking agent, etc. are not extracted from the impregnated polymer solution, and cross-linking is performed. Must be inert to the reaction. Specific examples thereof include water, alcohol and the like.

The cross-linking reaction is usually carried out at a temperature in the range of 5 to 90 ° C. over 1 to 15 hours. The temperature is preferably in the range of 30 to 90 ° C.

After the cross-linking reaction is completed, the generated particles are filtered off, and then washed with a hydrophilic organic solvent such as water, methanol, ethanol, etc. to remove unreacted polymers, suspension medium, etc., to obtain porous polymer particles. Particles are obtained in which at least a part of the surface is coated with a coating layer containing a crosslinked polymer having a hydroxyl group.

(Dextran graft)
Then, a group capable of reacting with dextran is introduced into the crosslinked polymer having a hydroxyl group. The method for introducing a group capable of reacting with dextran is not particularly limited, and a known method or the like can be used. For example, the epoxy group can be introduced by immersing the particles in an alkaline aqueous solution having a predetermined concentration to prepare a dispersion, and then adding an alkyl halide compound having an epoxy group to the dispersion to cause a reaction. Examples of the alkaline aqueous solution include an aqueous solution of sodium hydroxide. In addition, examples of the alkyl halide compound having an epoxy group include epichlorohydrin.

Then, the particles into which the group capable of reacting with dextran is introduced are dispersed in the aqueous solution of dextran, and then a catalyst or the like is added to react the group capable of reacting with dextran and dextran. Examples of the catalyst include an aqueous sodium hydroxide solution.

After completion of the reaction, the generated particles are filtered off, washed, and unreacted polymers are removed. Then, at least a part of the surface of the porous polymer particles is grafted with a crosslinked polymer having a hydroxyl group by dextran. A separator coated with a coating layer containing the graft polymer is obtained.

The amount of the coating layer is preferably 30 to 400 mg, more preferably 50 to 400 mg, and even more preferably 100 to 400 mg per 1 g of the porous polymer particles. When the ratio of the coating layer is 400 mg or less with respect to 1 g of the porous polymer particles, the coating layer can be made into a thin film, and the liquid permeability when used as a column tends to be further improved. Further, when the ratio of the coating layer is 30 mg or more with respect to 1 g of the porous polymer particles, the amount of protein adsorbed tends to be further increased. The amount of the coating layer can be measured by the weight loss of thermal decomposition, the Anthrone method or the like.

(Introduction of ion exchange group)
The separating material provided with the coating layer can be used for ion exchange purification, affinity purification, etc. by introducing an ion exchange group, a ligand (protein A), or the like via a hydroxyl group or the like on the surface. Examples of the method for introducing an ion exchange group include a method using an alkyl halide compound.

Examples of the alkyl halide compound include a monohalogenocarboxylic acid and its sodium salt, a primary or secondary or tertiary amine having at least one alkyl halide group, a hydrochloride thereof, and a quaternary having at least one alkyl halide group. Examples include ammonium salts. Examples of the monohalogenocarboxylic acid include monohalogenoacetic acid and monohalogenopropionic acid. Examples of the tertiary amine having at least one alkyl halide group include diethylaminoethyl chloride. These alkyl halide compounds are preferably bromides or chlorides. The amount of the alkyl halide compound used is preferably 0.2% by mass or more with respect to the total mass of the separating material to which the ion exchange group is imparted.

For the introduction of the ion exchange group, it is effective to use an organic solvent in order to promote the reaction. Examples of the organic solvent include alcohols such as ethanol, 1-propanol, 2-propanol, 1-butanol, isobutanol, 1-pentanol and isopentanol.

Normally, the ion exchange group is introduced into the hydroxyl group on the surface of the separating material. Therefore, the wet particles are drained by filtration or the like, immersed in an alkaline aqueous solution having a predetermined concentration, left for a certain period of time, and then water-organic. In a solvent mixing system, the above alkyl halide compound is added and reacted. Examples of the alkaline aqueous solution include an aqueous solution of sodium hydroxide. This reaction is preferably carried out at a temperature of 40 to 90 ° C. under reflux for 0.5 to 12 hours. The type of alkyl halide used in the above reaction determines the applied ion exchange group.

As a method for introducing an amino group which is a weakly basic group as an ion exchange group, at least one of the above alkyl halide compounds is substituted with an alkyl halide group, mono-, di -Or a method of reacting tri-alkylamine, mono-alkyl-mono-alkanolamine, di-alkyl-mono-alkanolamine, mono-alkyl-di-alkanolamine, etc., or at least one of the alkanol groups is halogenated. Methods for reacting mono-, di- or tri-alkanolamines, mono-alkyl-mono-alkanolamines, di-alkyl-mono-alkanolamines, mono-alkyl-di-alkanolamines, etc. substituted with alkanol groups, etc. Can be mentioned. The amount of these alkyl halide compounds used is preferably 0.2% by mass or more with respect to the total mass of the separating material. The reaction conditions are preferably 40 to 90 ° C. and 0.5 to 12 hours.

As a method for introducing a quaternary ammonium group of a strongly basic group as an ion exchange group, first, a tertiary amino group is introduced, and the tertiary amino group is reacted with an alkyl halide group-containing compound such as epichlorohydrin, and 4 A method of converting to a quaternary ammonium group can be mentioned. Further, a quaternary ammonium halide such as quaternary ammonium chloride may be reacted with the separating material.

Examples of the method for introducing a carboxy group, which is a weakly acidic group, as an ion exchange group include a method in which a monohalogenocarboxylic acid such as monohalogenacetic acid or monohalogenopropionic acid or a sodium salt thereof is reacted as the alkyl halide compound. .. The amount of these alkyl halide compounds used is preferably 0.2% by mass or more with respect to the total mass of the separating material into which the ion exchange group is introduced.

As a method for introducing a sulfonic acid group, which is a strongly acidic group, as an ion exchange group, a glycidyl compound such as epichlorohydrin is reacted with a separating material, and a sulfite such as sodium sulfite or sodium bisulfite or a sulfite is obtained. A method of adding a separating material to the saturated aqueous solution of the above can be mentioned. The reaction conditions are preferably 30 to 90 ° C. for 1 to 10 hours.

On the other hand, as a method of introducing an ion exchange group, a method of reacting 1,3-propane sultone with a separating material in an alkaline atmosphere can also be mentioned. It is preferable to use 1,3-propane sultone in an amount of 0.4% by mass or more based on the total mass of the separating material. The reaction conditions are preferably 0 to 90 ° C. for 0.5 to 12 hours.

The separating material of the present embodiment is suitable for separation by electrostatic interaction of proteins and for affinity purification. For example, the separating material of the present embodiment is added to a mixed solution containing a protein, and only the protein is adsorbed on the separating material by electrostatic interaction, and then the separating material is filtered off from the solution to obtain a salt concentration. When added to a high aqueous solution, the protein adsorbed on the separating material can be easily desorbed and recovered. In addition, the separating material of the present embodiment can also be used in column chromatography.

As the biopolymer that can be separated using the separating material of the present embodiment, a water-soluble substance is preferable. Specifically, it is a protein such as blood protein such as serum albumin and immunoglobulin, an enzyme existing in the living body, a protein physiologically active substance produced by biotechnology, a DNA, and a biopolymer such as a peptide having physiological activity. Yes, preferably the molecular weight is 2 million or less, more preferably 500,000 or less. In addition, it is necessary to select the properties, conditions, etc. of the separating material according to the isoelectric point, ionization state, etc. of the protein according to a known method. As a known method, for example, the method described in JP-A-60-169427 can be mentioned.

In the separating material of the present embodiment, after forming a coating layer, an ion exchange group and protein A are introduced on the surface of the separating material, so that particles or synthetic particles made of natural polymers can be separated in the separation of biopolymers such as proteins. Each has the advantages of particles made of polymers. In particular, the porous polymer particles in the separating material of the present embodiment are obtained by the above-mentioned method, and therefore have durability and alkali resistance. Further, the separating material of the present embodiment is provided with a coating layer containing the above-mentioned graft polymer (graft polymer in which dextran is grafted on a crosslinked polymer having a hydroxyl group), thereby reducing non-specific adsorption and adsorbing and desorbing proteins. Tends to occur easily. Further, the separating material of the present embodiment tends to have a large adsorption amount (dynamic adsorption amount) of proteins and the like under the same flow velocity.

The liquid passing speed in the present specification represents the liquid passing speed when a stainless column having a diameter of 7.8 × 300 mm is filled with the separating material of the present embodiment and the liquid is passed through. When the separating material of the present embodiment is packed in a column, it is preferable that the liquid passing speed is 800 cm / h or more when the column pressure is 0.3 MPa. When separating proteins and the like by column chromatography, the flow rate of the protein solution and the like to be passed through the column is generally in the range of 400 cm / h or less. On the other hand, when the separating material of the present embodiment is used, a high adsorption amount can be maintained even if it is used at a liquid passing speed of 800 cm / h or more, which is faster than the conventional separating material for protein separation.

The average particle size of the separating material of the present embodiment is preferably 10 to 300 μm, and for use in preparative or industrial chromatography, 10 to 100 μm in order to avoid an extreme increase in column internal pressure. It is preferably 50 to 100 μm, and more preferably 50 to 100 μm.

When the separating material of the present embodiment is used as a column filler in column chromatography, it is excellent in operability because the volume change in the column is small regardless of the nature of the eluate used.

The porosity of the separating material is preferably 30% by volume or more and 70% by volume or less, and more preferably 40% by volume or more and 70% by volume or less based on the total volume of the separating material. The separating material preferably has macropores (macropores), and the pore diameter (mode diameter) of the separating material is preferably 0.1 to 0.5 μm. The pore size of the separating material is more preferably 0.2 to 0.5 μm. When the pore diameter is 0.1 μm or more, a substance tends to easily enter the pores, and when the pore diameter is 0.5 μm or less, the specific surface area becomes sufficient.

The specific surface area of the separating material ^{is preferably 30 m 2} / g or more. From the viewpoint of higher practicality, the specific surface area is ^{more preferably 35 m 2} / g or more, and further preferably 40 m ² / g or more. When the specific surface area is 30 m ² / g or more, the amount of the substance to be separated tends to be large.

In this embodiment, the separating material in the form of introducing an ion exchange group has been described, but it can be used as a separating material without introducing an ion exchange group. Such separators can be used, for example, in gel filtration chromatography.
Further, the column of the present embodiment includes the separating material of the present embodiment. The column of the present embodiment can be produced by filling the column with the separating material of the present embodiment. The method of filling the column with the separating material is not particularly limited, and for example, a known method can be adopted.

Hereinafter, the present invention will be described with reference to Examples, but the present invention is not limited to these Examples.

(Example 1)
<Synthesis of Porous Polymer Particle 1>
16 g of divinylbenzene (manufactured by Nippon Steel & Sumikin Chemical Co., Ltd., trade name "DVB960") having a purity of 96%, 6 g of Span80, and 0.64 g of benzoyl peroxide were added to a 500 mL three-necked flask to prepare a dispersed phase. Moreover, 0.5 mass% of polyvinyl alcohol aqueous solution was used as a continuous phase. After emulsifying the continuous phase and the dispersed phase using a microprocess server, the obtained emulsion was transferred to a flask and stirred using a stirrer for about 8 hours while heating in a water bath at 80 ° C. The obtained particles were filtered and then washed with acetone to obtain porous polymer particles 1. The particle size of the porous polymer particles 1 was measured with a flow-type particle size measuring device, and the average particle size and the particle size of C.I. V. The value was calculated. The results are shown in Table 1.

<Formation of coating layer>
4 g of sodium hydroxide and 0.4 g of glycidyl phenyl ether were added to 100 mL of an aqueous solution (2% by mass) of agarose (Mw: 120,000) and reacted at 70 ° C. for 12 hours to introduce a phenyl group into agarose. The obtained modified agarose was precipitated with isopropyl alcohol. The precipitated modified agarose was filtered off, dissolved in water again, and then the operation of precipitating the modified agarose with isopropyl alcohol was repeated three more times to obtain modified agarose. The obtained modified agarose was dissolved in water again to prepare a 20 mg / mL modified agarose aqueous solution. The modified agarose was adsorbed on the porous polymer particles 1 by adding 10 g of the porous polymer particles 1 to 700 mL of this aqueous solution and stirring at 55 ° C. for 24 hours. The porous polymer particles 1 on which the modified agarose was adsorbed were filtered and washed with hot water.

(Crosslinking)
The modified agarose adsorbed on the surface of the porous polymer particles 1 was crosslinked as follows. After preparing a dispersion in which 10 g of the porous polymer particles 1 were dispersed in 350 g of a 0.4 M sodium hydroxide aqueous solution, 10 g of ethylene glycol diglycidyl ether was added to the dispersion, and the mixture was stirred at room temperature for 12 hours. Modified agarose was crosslinked. The obtained particles were filtered off, and then washed with 2% by mass of a hot sodium dodecyl sulfate aqueous solution and then with pure water.

(Dextran graft)
Next, after preparing a dispersion in which 10 g of particles were dispersed in 350 g of a 0.4 M aqueous sodium hydroxide solution, 20 g of epichlorohydrin was added to the dispersion, and the mixture was stirred for 3 hours to crosslink the modified agarose. An epoxy group was introduced into the body. The obtained particles were washed with 2% by mass of a hot sodium dodecyl sulfate aqueous solution and then with pure water.

About 10 g of the obtained particles were put into 100 mL of a 10 mass% dextran (Mw: 40,000) aqueous solution and stirred at 25 ° C. for 1 hour to prepare a dispersion. Then, 100 mL of 1 M aqueous sodium hydroxide solution and 0.36 g of sodium borohydride were added to the dispersion, and the mixture was stirred at 25 ° C. for 18 hours and grafted with dextran.

(Evaluation of coating layer amount)
By thermogravimetric analysis, the amount of coating layer (mg) per 1 g of porous polymer particles was calculated.
The results are shown in Table 2.

(Graft amount evaluation)
The amount of graft (mg) per 1 g of porous polymer particles was calculated by thermogravimetric analysis of the particles before and after grafting. The results are shown in Table 2.

(Evaluation of non-specific adsorption of proteins)
0.5 g of the obtained separating material was put into 50 mL of phosphate buffer (pH 7.4) having a BSA (Bovine Serum Albumin) concentration of 20 mg / mL, and the mixture was stirred at room temperature for 24 hours. Then, centrifugation was performed to remove the supernatant. The amount of BSA adsorbed on the separating material was calculated from the BSA concentration in the supernatant obtained by measuring the absorbance at 280 nm of the supernatant with a spectrophotometer. The results are shown in Table 3.

<Introduction of ion exchange group>
Water was removed from the obtained particle dispersion by centrifugation. 20 g of the obtained particles were dispersed in an aqueous solution of diethylaminoethyl chloride hydrochloride (an aqueous solution obtained by dissolving 60 g of diethylaminoethyl chloride hydrochloride in water to make 100 g), stirring at 70 ° C. for 10 minutes, and then heating to 70 ° C. 100 mL of a 5 M aqueous sodium hydroxide solution was added and reacted for 1 hour. After completion of the reaction, the product was collected by filtration and washed twice with water / ethanol (volume ratio 8/2) to obtain a separator having a diethylaminoethyl (DEAE) group as an ion exchange group (DEAE modification). The pore diameter (mode diameter), specific surface area and porosity (porosity) of the obtained separating material were measured by a mercury intrusion method. The results are shown in Table 3.

(Ion exchange capacity evaluation)
0.2 to 0.3 g of the separating material swollen with water for 12 hours or more was quantified, transferred to a beaker, 20 mL of 0.1 N sodium hydroxide solution was added, and the mixture was stirred at 25 ° C. for 1 hour. Then, suction filtration was performed using a filter, and the particles on the filter were washed until the washing liquid became neutral. Then, the particles were transferred to a beaker, 20 mL of a 0.1 N hydrochloric acid aqueous solution was added, and the mixture was stirred at room temperature for 1 hour. Then, suction filtration was performed using a filter, and the particles on the filter were washed until the washing liquid became neutral. The ion exchange capacity (mmol / mL) of the separating material was determined by titrating this cleaning solution with a 0.1 N aqueous sodium hydroxide solution using an automatic potentiometric titrator. The results are shown in Table 3.

<Column characterization>
(Evaluation of liquid flow and evaluation of dynamic adsorption amount)
The obtained separating material was filled in a stainless steel column having a diameter of 7.8 × 300 mm as a slurry (solvent: methanol) having a concentration of 30% by mass for 15 minutes. Then, water was passed through the column while changing the flow velocity, the relationship between the flow velocity and the column pressure was measured, and the liquid flow velocity (linear flow velocity) at 0.3 MPa was measured. The results are shown in Table 3.

The amount of dynamic adsorption was measured as follows. A 20 mmol / L Tris-hydrochloric acid buffer (pH 8.0) was passed through the column by 10 column volumes. Then, a 20 mmol / L Tris-hydrochloric acid buffer having a BSA concentration of 2 mg / mL was passed through, and the BSA concentration at the column outlet was measured by UV measurement. The buffer was passed through the buffer until the BSA concentrations at the column inlet and outlet were matched, and the mixture was diluted with 1 M NaCl Tris-hydrochloric acid buffer in a volume of 5 columns. The amount of dynamic adsorption at 10% breakthrough was calculated using the following formula. The results are shown in Table 3.
q ₁₀ = c _f F (t ₁₀ −t ₀ ) / V _B
q ₁₀ : Dynamic adsorption amount at 10% breakthrough (mg / mL wet resin)
cf: Injecting BSA concentration F: Flow velocity (mL / min)
V _B : Bed volume (mL)
time at t ₁₀ : 10% breakthrough (min)
t ₀ : BSA injection start time (min)

(Example 2)
A separating material was prepared in the same manner as in Example 1 except that the Mw of the dextran to be grafted was changed to 150,000, and evaluated in the same manner as in Example 1.

(Example 3)
A separating material was prepared in the same manner as in Example 1 except that the Mw of the dextran to be grafted was changed to 500,000, and evaluated in the same manner as in Example 1.

(Example 4)
A separating material was prepared in the same manner as in Example 1 except that the Mw of the dextran to be grafted was changed to 1,000,000, and evaluated in the same manner as in Example 1.

(Comparative Example 1)
The porous polymer particles 1 were used as they were as a separating material and evaluated in the same manner as in Example 1.

(Comparative Example 2)
A separating material was prepared in the same manner as in Example 1 except that polyvinyl alcohol (PVA) having an Mw of 12000 was grafted instead of dextran, and evaluated in the same manner as in Example 1.

(Comparative Example 3)
A separating material was prepared in the same manner as in Example 1 except that Mw was grafted with 22000 agarose instead of dextran, and evaluated in the same manner as in Example 1.

(Comparative Example 4)
A separating material was prepared in the same manner as in Example 1 except that Mw was grafted with 21000 methylcellulose instead of dextran, and evaluated in the same manner as in Example 1.

(Comparative Example 5)
Commercially available agarose particles (Capto DEAE: GE Healthcare) (hereinafter referred to as “porous polymer particles 2”) were used as they were as a separating material and evaluated in the same manner as in Example 1.

(Comparative Example 6)
<Synthesis of Porous Polymer Particles 3>
Same as porous polymer particles 1 except that 11.2 g of 2,3-dihydroxypropyl methacrylate and 4.8 g of ethylene glycol dimethacrylate were used instead of 16 g of divinylbenzene, and the amount of Span80 was changed from 6 g to 5 g. Porous polymer particles 3 were obtained by the above method. The particle size of the porous polymer particles 3 was measured with a flow-type particle size measuring device, and the average particle size and the particle size of C.I. V. The value was calculated. The results are shown in Table 1.

<Formation of coating layer>
In 5 g of distilled water, 1 g of dextran (Mw: 150,000), 0.6 g of sodium hydroxide and 0.15 g of sodium borohydride were dissolved. 4 g of the porous polymer particles 3 were mixed with 6 g of the obtained solution, and dextran was adsorbed on the porous polymer particles 3. The porous polymer particles 3 on which dextran was adsorbed were added to 1 L of a mixed solution of ethyl cellulose and toluene (ethyl cellulose concentration: 1% by mass) and stirred to disperse the particles to obtain a suspension. 5 mL of epichlorohydrin was added to the obtained suspension, the temperature was raised to 50 ° C., and the mixture was stirred at this temperature for 6 hours to crosslink the dextran adsorbed on the surface of the porous polymer particles 3. After completion of the reaction, the particles were separated from the suspension and washed successively with toluene, ethanol and distilled water to prepare a separating material. The obtained separating material was evaluated in the same manner as in Example 1.

From the results in Table 3, it can be seen that the separating materials of Examples 1 to 4 have reduced non-specific adsorption of proteins, a high adsorption amount, and excellent liquid permeability when used as a column.

Claims (6)

It comprises a porous polymer particle and a coating layer that covers at least a part of the surface of the porous polymer particle.
The porous polymer particles contain a polymer having a structural unit derived from a monoma containing divinylbenzene.
The coating layer contains a graft polymer in which dextran is grafted on a crosslinked polymer having a hydroxyl group.
The crosslinked polymer having a hydroxyl group, Ri modified product der modified by hydrophobic groups, Ru Oh crosslinked polymer derived from the denatured product of the agarose, the separating material. The separating material according to claim 1, wherein the pore diameter (mode diameter) is 0.1 to 0.5 μm. The separating material according to claim 1 or 2 , wherein the coefficient of variation of the particle size of the porous polymer particles is 5 to 15%. The separating material according to any one of claims 1 to 3 , wherein the weight average molecular weight of the dextran is 40,000 to 1,000,000. The separating material according to any one of claims 1 to 4 , comprising the coating layer of 30 to 400 mg per 1 g of the porous polymer particles. A column comprising the separating material according to any one of claims 1 to 5.